let-7: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - let-7

Synonyms -

Cytological map position -

Function - post-transcriptional gene silencing

Keywords - post-transcriptional gene silencing, metamorphosis

Symbol - let-7

FlyBase ID: FBgn0262406

Genetic map position -

Classification - microRNA

Cellular location - cytoplasmic



NCBI link: Entrez Gene
Recent literature
Ma, Q., de Cuevas, M. and Matunis, E.L. (2016). Chinmo is sufficient to induce male fate in somatic cells of the adult Drosophila ovary. Development [Epub ahead of print]. PubMed ID: 26811385
Summary:
Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (DsxM) to maintain the male identity of somatic cyst stem cells and their progeny. This study reports that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a DsxM-independent mechanism. In contrast, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by loss of the canonical female sex determinant DsxF, indicating that chinmo acts together with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss of function phenotype, but no apparent phenotypes were found upon removal of let-7 in the adult ovary or testis. The finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis.

Chawla, G., Deosthale, P., Childress, S., Wu, Y. C. and Sokol, N. S. (2016). A let-7-to-miR-125 MicroRNA switch regulates neuronal integrity and lifespan in Drosophila. PLoS Genet 12: e1006247. PubMed ID: 27508495
Summary:
Messenger RNAs (mRNAs) often contain binding sites for multiple, different microRNAs (miRNAs). However, the biological significance of this feature is unclear, since such co-targeting miRNAs could function coordinately, independently, or redundantly with one another. This study shows that two co-transcribed Drosophila miRNAs, let-7 and miR-125, non-redundantly regulate a common target, the transcription factor Chronologically Inappropriate Morphogenesis (Chinmo). Novel adult phenotypes were characterized that were associated with loss of both let-7 and miR-125, which are derived from a common, polycistronic transcript that also encodes a third miRNA, miR-100. Consistent with the coordinate upregulation of all three miRNAs in aging flies, these phenotypes include brain degeneration and shortened lifespan. However, transgenic rescue analysis reveal separable roles for these miRNAs: adult miR-125 but not let-7 mutant phenotypes are associated with ectopic Chinmo expression in adult brains and are suppressed by chinmo reduction. In contrast, let-7 is predominantly responsible for regulating chinmo during nervous system formation. These results indicate that let-7 and miR-125 function during two distinct stages, development and adulthood, rather than acting at the same time. These different activities are facilitated by an increased rate of processing of let-7 during development and a lower rate of decay of the accumulated miR-125 in the adult nervous system. Thus, this work not only establishes a key role for the highly conserved miR-125 in aging, it also demonstrates that two co-transcribed miRNAs function independently during distinct stages to regulate a common target, raising the possibility that such biphasic control may be a general feature of clustered miRNAs.
BIOLOGICAL OVERVIEW

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA (Lagos-Quintana, 2001) is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether Drosophila let-7 expression is regulated by ecdysone, Northern blot analysis was used to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. Ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects (Sempere, 2002).

The temporal coordination of cell proliferation, differentiation, and apoptosis during development is essential for the correct morphogenesis of an adult animal. In the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, genetic regulatory circuits control the timing of the transition from larval to adult stages. In Drosophila, larvae pupariate and initiate metamorphosis after the third larval instar. In C. elegans, the adult stage follows immediately after the fourth (final) larval molt. Studies on metamorphosis in Drosophila have revealed a pivotal role for the steroid hormone 20-hydroxyecdysone (ecdysone) and ecdysone-regulated gene expression. Ecdysone orchestrates a complex, hierarchical gene expression cascade that transforms the larva into a highly motile reproductive adult fly. In C. elegans, the heterochronic gene pathway generates the temporal contexts in which the appropriate developmental programs are executed throughout development of the larva to the adult. Although nematodes do not undergo an overt metamorphosis, as does Drosophila, the life cycle of the two animals is similar in that both undergo stages of molting development prior to the adult stage. This common developmental strategy groups them together as ecdysozoans, in recognition that a common ancestor underwent a series of larval stages punctuated by cuticular molts (Sempere, 2002 and references therein).

Dynamic changes in ecdysone levels regulate progression through the larval stages of holometabolous insects. During the third (final) instar of Drosophila, a series of low-level ecdysone pulses signal the transition from feeding to wandering, in preparation for pupariation. Following a high-level ecdysone pulse, the white prepupa forms and larval tissues begin to either remodel or histolize. Meanwhile, precursors of adult structures and tissues, which include the imaginal discs, histoblasts, and imaginal cell nests along the midgut, initiate their proliferation and differentiation programs. Some 10-12 h after puparium formation, a second ecdysone pulse leads to head eversion and pupa formation. A broad and high-level peak of ecdysone secretion during the pupal stage triggers the terminal differentiation of the adult structures (Sempere, 2002).

Ecdysone pulses trigger each developmental transition by initiating a program of downstream gene expression. Ecdysone binds a heterodimeric protein receptor, composed of an Ecdysone receptor subunit (EcR) and an RXR-like subunit encoded by the ultraspiracle (usp) gene. The ecdysone-receptor complex binds to a cis-acting regulatory element, known as the ecdysone response element (EcRE) in the enhancers of specific target genes, thereby causing an increase in target gene transcription. According to the Ashburner model (Ashburner, 1974), postlarval development begins by the hormone-dependent activation of a small set of 'early' genes that include Broad-Complex (BR-C), E74, and E75. Each of these genes encodes a complex set of protein isoforms that function as sequence-specific DNA binding proteins and transcriptional regulators. The protein products of early genes activate a second cascade of gene expression, the 'late' genes, and inhibit early gene expression by feedback. The outcome of this unfolding genetic cascade is manifest at the cell and tissue levels as biochemical and morphological differentiation (Sempere, 2002 and references therein).

In C. elegans, a newly hatched larva develops through four larval stages (L1 to L4), punctuated by molts, to a reproductive adult. Blast cells, set aside during embryogenesis, divide during the larval stages and give rise to stage-specific larval features, and the adult-specific reproductive structures. The heterochronic genes lin-4 and let-7 are crucial for promoting the transitions from early to late developmental programs (Lee, 1993; Reinhart, 2000). lin-4 and let-7 are small regulatory RNAs (22 and 21 nucleotides, respectively), which act as translational repressors by base-pairing with the 3'-UTRs of their target gene mRNAs (Lee, 1993; Reinhart, 2000). The accumulation of lin-4 RNA at the beginning of the L2 stage downregulates the protein levels of its target genes, lin-14 and lin-28, and permits the coordinated transition from L1 to later programs. Similarly, the accumulation of let-7 RNA at the beginning of the L4 stage downregulates lin-41 (Slack, 2000) and possibly lin-57 (A. Rougvie, personal communication to Sempere, 2002) and promotes the larval to adult (L/A) transition from L4 to adult programs (Sempere, 2002 and references therein).

The participation of two small regulatory RNAs in the heterochronic pathway raises the question of whether similar regulatory RNAs could be involved in the control of postembryonic development in other animals. Indeed, let-7 is conserved across the bilaterian clade, including flies and humans (Pasquinelli, 2000). In invertebrates, let-7 RNA expression coincides with the onset of the L/A transition. Similarly, let-7 expression is upregulated during vertebrate development, although at somewhat different stages depending on the species (Pasquinelli, 2000). Studies in human (Pasquinelli, 2000) and murine cell lines demonstrate the presence of high levels of let-7 RNA in mature cell types (e.g., brain and lung) and marginal let-7 expression in immature or totipotent cell types (bone marrow and murine embryonic stem cell). Taken together, these observations suggest a general role for let-7 in the terminal differentiation of bilaterians (Pasquinelli, 2000, Sempere, 2002).

In Drosophila, let-7 RNA first appears at the end of the third larval instar, a few hours before puparium formation, and reaches high levels during pupal development (Pasquinelli, 2000; Hutvagner, 2001). Given the leading role of ecdysone in the temporal coordination of metamorphosis, investigations were carried out to see whether the expression of let-7 in Drosophila is dependent on the ecdysone gene pathway. Indeed, ecdysone and the early ecdysone-inducible gene BR-C are required for let-7 expression in intact animals, organ cultures, and S2 cells. These results suggest that hormone-induced expression of let-7 could control developmental stage transitions in animals (Sempere, 2002).

Several lines of evidence are presented that both ecdysone and the early ecdysone-inducible gene BR-C are required for the expression of let-7 RNA in Drosophila. This indicates that the ecdysone pathway regulates the temporal expression of let-7 in the fly. Previously identified ecdysone-inducible genes include a series of transcriptional factors that are organized in a hierarchical network to control metamorphic processes. In C. elegans, let-7 is a translational repressor (Slack, 2000), and so Drosophila let-7 may mediate aspects of the hormonal control of metamorphosis by regulating gene expression post-transcriptionally (Sempere, 2002).

The primary evidence that let-7 expression is triggered by the ecdysone pathway comes from two lines of experiments: (1) mutant animals defective in ecdysone biosynthesis or in BR-C activity display reduced or absent let-7 RNA levels; (2) sustained expression of let-7 RNA in organ culture requires the application of exogenous ecdysone. These experiments do not rule out the possibility that let-7 expression could be a collateral consequence of ecdysone signals, for example, as a consequence of pupariation or progression through metamorphosis. However, if this were the case, then ecdysone-induced let-7 expression would not be expected in S2 cells, since S2 cells do not undergo morphogenesis. The finding that ecdysone induces let-7 expression in S2 cells strongly suggests that ecdysone pathway triggers let-7 expression within the cells exposed to the hormone, and independently of overt metamorphosis. The requirement of BR-C activity in animals and S2 cells for let-7 RNA induction indicates that BR-C is an intermediate player between the ecdysone signal and activation of let-7, and that the let-7 response to ecdysone exhibited in S2 cells likely reflects the same process as in vivo. One difference between the response to ecdysone in S2 cells and animals is that, in S2 cells, let-7 expression begins about 24 h after the addition of ecdysone, while in animals, let-7 expression begins about 4 h after the pulse of ecdysone at the end of the third larval stage. Components of the pathway mediating let-7 activation by ecdysone in S2 cells may be relatively limiting compared to intact animals, and perhaps the concentration of ecdysone needed to activate let-7 expression when applied to S2 cells may not be as effective as that in vivo. Although a role for RNA stability or processing in the induction of let-7 by ecdysone cannot be ruled out, the simplest interpretation is that let-7 induction occurs at the transcriptional level, since levels of let-7 RNA and its precursor let-7L (Grishok, 2001; Hutvagner, 2001) increase or decrease coordinately, depending on the status of ecdysone signaling (Sempere, 2002).

The timing of let-7 expression is conserved in invertebrates: let-7 RNA accumulates toward the end of larval development in flies, worms, and mollusks, coinciding with the specification of adult programs (Pasquinelli, 2000). Although the results indicate that in dipterans let-7 is induced by ecdysone, let-7 expression could be coupled to other signals in other animals. In C. elegans, the timing of let-7 expression is controlled partly by upstream components of the heterochronic pathway in conjunction with other regulatory signals. daf-12, an orphan nuclear hormone receptor (NHR), is an upstream component of the heterochronic pathway implicated in the regulation of let-7 expression. DHR96, the closest ortholog of daf-12 in Drosophila, also encodes an orphan NHR. DHR96 is one of the eight NHRs whose expression is regulated by ecdysone during metamorphosis. daf-12 and dhr96 could represent an evolutionarily conserved point of convergence in C. elegans and Drosophila let-7 regulatory pathways. Whether C. elegans utilizes other components of the ecdysone pathway during its development remains an open question. More than 200 NHRs have been predicted in C. elegans, and some of these are clear orthologs of Drosophila NHRs involved in metamorphosis, suggesting that they could play similar roles in C. elegans (Sempere, 2002).

The increase in let-7 RNA at pupariation in response to ecdysone/BR-C activity has suggested a potential role for let-7 in Drosophila metamorphosis. In C. elegans, let-7 promotes the larval to adult transition by downregulating lin-41 protein levels. There are several complementary sites to let-7 in the 3'-UTR of lin-41 mRNA to which let-7 could bind to repress translation (Slack, 2000). lin-41 codes for a RBCC protein (Slack, 2000) and is a founding member of the NHL domain family. In Drosophila, there are three orthologs of lin-41: dappled, brat, and mei-P26. These lin-41 orthologs appear to be involved somehow in growth suppression in flies; mutations in dappled and brat result in melanotic tumors, and mutations in mei-P26 result in ovarian tumors, dappled and brat are expressed at the end of the third instar in fat bodies and ring gland (brain), and brain and wing imaginal discs, respectively. mei-P26 is expressed in the germ line. These expression patterns overlap with that of let-7 RNA, suggesting that the lin-41 orthologs could be let-7 targets. Indeed, sequences in the 3'-UTR of dappled, brat, and mei-P26 resemble let-7 complementary sites to which let-7 could bind. Future work is required to determine whether let-7 is a regulator of these and/or other genes and to assess the implications of this regulation in metamorphic processes, such as apoptosis, differentiation, and morphogenesis (Sempere, 2002 and references therein).


RNA structure

Bases - 70 (precursor) and 21 (biologically active form)

Structural Domains

The gene products of Drosophila and C. elegans let-7 are two noncoding small RNAs of 21 (let-7S, short) and approximately 70 (let-7L, long) nucleotides. let-7S is the biologically active isoform (Reinhart, 2000), which results from the processing of the precursor let-7L (Grishok, 2001; Hutvagner, 2001; Lagos-Quintana, 2001). (Sempere, 2002).


let-7: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 10 June 2002

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